Abstract

A strain of Saccharomyces cerevisiae (R57) highly tolerant to inhibitors found in the lignocellulosic substrate hardwood spent sulfite liquor (HWSSL) was created through genome shuffling. The goal of my research was to identify the mutations responsible for the tolerance phenotype. The genomes of R57 and wild type strain (CEN.PK 113-7D) were previously sequenced and compared for mutations (Dominic Pinel unpublished results). Forty-seven single nucleotide polymorphisms (SNPs) predicted by genome sequence comparison were PCR amplified from R57 genomic DNA for confirmation by Sanger sequencing. Of the 47, 20 were confirmed affecting 17 genes. These SNP-containing regions were amplified from mixed populations of cells from the UV mutagenesis treatment and rounds 1, 3 and 5 of the evolutionary engineered pool of cells. These amplicons were sequenced using the Ion Torrent sequencer revealing the frequencies of each SNP from each pool of cells as they progressed through the evolution. Mutated regions amplified from isolated colonies taken from the growth frontier of HWSSL gradient plates for the UV mutagenized and round 5 pools were sequenced using Sanger sequencing to reveal specific combinations of SNPs. The homozygous UBP7 c.2466T>A mutation was reconstituted in a diploid wild type background strain and tested against the diploid wild type and R57 strains. The UBP7 mutant showed increased tolerance to HWSSL. The combination of this data led to the hypothesis that of the 17 directly affected genes, 6 are more likely to play a role in the tolerance phenotype observed in R57 (UBP7, NRG1, MAL11, GDH1, GSH1 and ART5).